In order to understand cell growth of both normal and abnormal cells, scientists have sought to grow cells in chemically defined culture media. Nearly all animal cells in culture require serum for growth and serum is added to nutrient media. However, serum is a complex material containing many substances. The non-nutrient entities present in serum which promote cell growth have been termed "growth factors." Some growth factors have specific tissues as their target while others cause growth of broader spectrum of cell types. Numerous scientists have sought means to isolate and purify factors responsible for cell growth. Although some factors are chemically identifiable as steroid or polyamine in nature, most of the substances are polypeptide or protein in nature. Isolation and purification of these low concentrations of proteins in complex systems by present techniques are extremely difficult.
A technique employed and reported frequently in the prior art is protein fractionation employing various precipitating agents such as ethanol (R. S. Chang et al., Proc. Soc. Exptl. Biol. & Med., 102(1):213-217 (1959); H. Katsuta et al., Jpn. J. Exp. Med., 29:297-309 (1959)), zinc (R. S. Chang et al., loc. cit.) and ammonium sulfate (J. Michl, Exp. Cell. Res. 23:324-334 (1961); H. Katsuta et al, loc. cit.). This technique is cumbersome, usually involving many steps and not infrequently causing denaturation of some of the protein. A sequential combination of different techniques has also been employed. Thus, for example, R. W. Pierson, Jr. et al., J. Cell. Physiol., 79:319-330 (1972), reported a three technique procedure: chromatography, gel-filtration and polyacrylamide gel electrophoresis; R. Hoffman et al., Exp. Cell. Res. 85, 275-280 (1973), described a multi-technique procedure which include the techniques of ammonium sulfate precipitation, differential flotation, chromatographic separation, gel filtration, immunodiffusion and electrophoresis; and I. Lieberman et al., J. Biol. Chem., 233 (3):637-642 ( 1958) described a multitechnique procedure which include ammonium sulfate precipitation, twofold ethanol precipitation and chromatography on diethylaminoethylcellulose. Each technique of the multi-technique procedure generally requires several steps rendering these methods cumbersome. A substantially single technique separation, namely, paper curtain electrophoresis was employed by R. Holmes et al., J. Biophys. Biochem. Cytol. 10:389-401 (1961); however, this method is not very suitable for large scale preparation. Also H. Katsuta et al., loc. cit. used zone electrophoresis for separation but this method is less adaptable for large scale preparation. Although H. N. Antoniades et al., Endocrinology: 70:95-98 (1962) have reported an isolation of insulin-like complex (different material) from serum using chromatographic technique with a cation exchange resin, when that technique was used subsequently by Pierson et al., loc cit, for growth factor isolation, the isolated material retained only a minor part of the activity. This is illustrative of the problems encountered for as pointed out in the review article by D. Gospodarowicz et al., Ann. Rev. Bioch. 45, 531-558 (1976) during isolation, growth factors with additive or synergistic effect may be separated resulting in fractions that are much less active individually. A further difficulty pointed out is that the mitogen might account for only from 5.times.10.sup.-6 to 2.times.10.sup.-2 percent of the protein in the serum.
It is seen that the heretofore published methods are cumbersome, requiring a combination of techniques, or are not economically feasible or adaptable to a moderate or large scale operation, or do not provide a method for isolating the growth material without inactivation. It is desirable to have a simpler method for obtaining serum growth factors freed of many of the non-growth promoting substances. It is further desirable for any cell culture work to have a purified growth factors preparation instead of whole serum with its complex and varying composition.
In addition, there are not only many areas of practical application in which there may be made beneficial substitution of purified serum growth materials for whole serum, but the availability of growth material would make possible new areas of application of cell growth regulation. Some of the areas in which the availability of purified serum growth materials would be highly advantageous include in the study and cure of neoplastic diseases, dwarfism, and mental development (nerve growth factor), in the study of cellular immunity, in replacing serum for virus vaccine production, and for biomolecules production (e.g. urokinase or interferon production), in tissue grafting in burn cases, in animal husbandry for increasing and maintaining rapid growth, and wherever serum is required for tissue growth. Thus, it is seen that a purified growth material preparation is desirable for many applications.